Hematological and plasma profiles and ticks and tick-borne pathogens in wild Formosan black bears (Ursus thibetanus formosanus)

Background The endangered Formosan black bear (Ursus thibetanus formosanus) is the largest native carnivorous mammal in Taiwan. Diseases, poor management, illegal hunting, and habitat destruction are serious threats to the survival of bear populations. However, studies on the impact of diseases on bear populations are limited. Therefore, this study aimed to establish a database of the hematological and plasma profiles of free-ranging Formosan black bears and investigate the occurrence of ectoparasites, blood parasites, and vector-borne pathogens. Methods Formosan black bears were captured in Yushan National Park (YNP) and Daxueshan Forest Recreation Area (DSY) in Taiwan. Blood samples were collected from each bear for hematological analysis and plasma biochemistry using a hematology analyzer. Parasites and pathogens were detected using a thin blood smear with Wright–Giemsa staining and polymerase chain reaction (PCR) assay. Additionally, macroscopic ectoparasites were collected from bears to detect blood parasites and other pathogens. Moreover, the relationships between the bear variables (sex, age, and occurrence of parasites or pathogens), ectoparasites, and infectious agents were also analyzed. Results In all, 21 wild bears (14 in YNP and 7 in DSY) were captured and released during the satellite tracking studies. Hematological analysis and plasma biochemistry indicated significant differences in white blood cells (WBC), segments, creatine kinase (CK), and lactate dehydrogenase (LDH) levels between foot snare and culvert-captured bears. Additionally, there were significant differences in total plasma protein (TPP), creatinine, Ca2+, Mg2+, and K+ levels between male and female bears. Moreover, pathogen-infected bears had significantly higher erythrocyte sedimentation rate (ESR; 30 min and 1 h) and globulin levels than uninfected bears. In total, 240 ticks were collected from 13 bears, among which eight adult tick species were identified, including Haemaphysalis flava, Haemaphysalis hystricis, Amblyomma testudinarium, Ixodes ovatus, Dermacentor taiwanensis, Haemaphysalis longicornis, Ixodes acutitarsus, Amblyomma javanense, and nymphs belonging to Haemaphysalis spp. PCR revealed that 13 (61.90%) and 8 (38.10%) bears harbored Hepatozoon ursi and Babesia DNA, respectively. Among the ticks examined, 157 (65.41%) and 128 (53.33%) samples were positive for H. ursi and Babesia, respectively. Conclusions To the best of our knowledge, this is the first study to establish a database of the hematological and plasma profiles of wild Formosan black bears and investigate ectoparasite infestation and Hepatozoon and Babesia spp. infection. In conclusion, these findings may serve as a reference for monitoring the health and population of locally endangered bears. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s13071-024-06320-7.


Background
Large carnivores perform vital ecological functions increasing their ecological integrity and biodiversity.The Formosan black bear (Ursus thibetanus formosanus) is a subspecies of Asiatic black bear and is indigenous to Taiwan.Presently, the Formosan black bear is classified as "Vulnerable" by the International Union for Conservation of Nature (IUCN) Red List of Threatened Species and listed in Additional file 1 of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES).Additionally, the Formosan black bear is the largest native carnivore in Taiwan, and is listed as an endangered species under Taiwan's Wildlife Conservation Act.Notably, Formosan black bears function as umbrella and landscape species for conservation because of their wide home range [1].
Importantly, the conservation of bear populations needs to be considered in relation to several aspects, such as population structure, environment, genetics, disease, and management conditions.Habitat destruction and illegal hunting are critical threats to the survival of Formosan black bears [2].Recent ecological studies on carnivores have shown that epidemic diseases seriously threaten several endangered wild animals, especially in populations with reduced numbers, malnutrition, and stress, or in inbreeding groups.However, little is known about the effects of diseases on the population of Formosan black bear.To prevent further decline in the already small bear population, research should focus on potentially threatening diseases and environmental conditions [3].
Blood and plasma biochemical reference values can assist clinical veterinarians in interpreting individual nutritional and physiological conditions and serve as the basis for disease diagnosis.For example, a comprehensive understanding of the physiology, hematology, and serum biochemistry of American black bears (Ursus americanus) and brown bears (Ursus arctos) is crucial for evaluating population health managing both captive and wild bear populations [4][5][6][7][8].Additionally, serological studies have detected several pathogens in bears in America and Europe [9][10][11][12].However, information on the hematology and serum biochemistry of Asiatic black bears is limited, except for a few studies on captive bears in Thailand, Taiwan, and South Korea [13][14][15].
A comprehensive understanding of pathogen exposures is important to assess the overall health of bear populations and reveal possible infectious agents that may affect the health of humans and other animals.Blood macro-and micro-parasites, such as heartworms, Hepatozoon ursi, and Babesia spp., have been detected in the blood samples of different bear species in several countries [16][17][18][19][20]. Zoonotic pathogens, including Bartonella spp., Rickettsia spp., and Borrelia burgdorferi s.l., have also been detected in bear populations [21,22].Considering that wild bears can act as reservoirs of various pathogens, some of which pose risks to human health via zoonotic transmission, identifying these pathogens may help prevent potential disease threats to human populations.Among ectoparasites, lice (Trichodectes pinguis) and ticks (Haemaphysalis megaspinosa) were found on a gunshot-wounded Asiatic black bear [23].Additionally, Demodex ursi and various species of hard ticks (Amblyomma americanum, Amblyomma maculatum, Dermacentor variabilis, Ixodes scapularis, Ixodes affinis) were found on American black bears [24][25][26].
Although some physiological data are available [5,6,8,27,28], knowledge of potential infectious and vectorborne pathogens in Formosan black bears is still lacking.Therefore, this study aimed to establish a database of the hematological and plasma profiles of free-ranging Formosan black bears and investigate the occurrence of ectoparasites, blood parasites, and vector-borne pathogens.Overall, it is anticipated that this study will provide useful information for the conservation and management of endangered Formosan black bear populations.

Capture-release procedures for Formosan black bears
Formosan black bears were captured from November 2014 to July 2021 at Yushan National Park (YNP) and Daxueshan Forest Recreation Area (DSY) in Taiwan for Global Positioning System (GPS) telemetry.YNP is located in the Central Mountain Range (23° 24′ 26.0604″ N, 121° 1′ 17.3604″ E), which covers an area of 1031.214km 2 (Fig. 1).DSY is located in the southeastern part of the Xueshan Mountain Range, near Taichung City (24° 15′ 11.16″ N, 121° 0′ 31.68″E), covering an area of 39.63 km 2 (Fig. 1).Notably, these areas have a relatively high bear density.All procedures involving bear capture, release, anesthetization, and sampling have been approved by the Forestry and Nature Conservation Agency of the Ministry of Agriculture under the Agreement of the Use of Conserved Wild Animals (No. and Babesia spp.infection.In conclusion, these findings may serve as a reference for monitoring the health and population of locally endangered bears.Black bears were captured using an Aldrich springactivated foot snare [30] and culvert traps (L × W × H: 200 × 78 × 78 cm) [31] (Fig. 2a, b), with the former only applied in YNP where some sites had no road access and required a 3-day hike.Notably, both traps were used from November 2014 to May 2016, and only culverts were used afterwards.Captured bears were immobilized using mixed doses of different anesthetics based on the capture procedure and the individual bear status.For snare-captured and anxious bears, 0.03 mg/kg of dexmedetomidine (Dexdomitor ® , Pfizer Inc., New York, NY) and 3.0 mg/ kg of tiletamine-zolazepam (Zoletil ® , Virbac Inc., Carros, France) were used.For small bears captured in Culvert traps, 0.025 mg/kg of dexmedetomidine and 2.5 mg/ kg of tiletamine-zolazepam were used [32,33].Mixed anesthetics were delivered intramuscularly through blow darts.
After anesthetization, physical signs of anesthetic depth and physiological parameters, including body temperature and respiratory rate, were monitored and recorded.Pulse rate and blood oxygen saturation were monitored using a pulse oximeter (9847 V; NONIN Medical, Plymouth, MN, USA) with a sensor attached to the tongue.Individual sex, age, body weight, and morphometric characteristics were measured and recorded (Fig. 2c, d) [34].Bear age was estimated and classified as adult or subadult on the basis of first premolar tooth sections or tooth wear [34,35].Bears aged 4 years or older were classified as adults [36].After collection of blood samples and infesting ectoparasites, radio frequency identification (RFID) microchips were implanted into the hind necks of the bears, and ear tags were applied for individual identification.Additionally, GPS collars were attached to each bear for satellite tracking.At the end of the procedure, 10 mg of atipamezole (Antisedan ® , Pfizer Inc., New York, NY) for each mg of dexmedetomidine were administered intramuscularly to reverse the anesthetic effect [37].

Sample collection procedures
Blood samples (10-20 mL) were collected from the cephalic or femoral vein in ethylenediaminetetraacetic acid (EDTA) anticoagulant tubes (BD Biosciences, Franklin Lakes, NJ, USA) and stored at 4 °C in a portable refrigerator.Ectoparasites were collected from the skin using tweezers and stored in microcentrifuge tubes containing 70% ethanol [38].All samples were transported within 3 days to the Veterinary Medical Teaching Hospital and the Laboratory of Public Health and Epidemiology, National Pingtung University of Science and Technology for morphological identification and laboratory processes.

Morphological identification of ectoparasites
The ectoparasites collected in this study were all ticks.Briefly, ticks were surface-rinsed twice with deionized water and observed under a dissecting microscope for morphological identification using taxonomic keys [40,41].After morphological identification, each tick was individually placed in a microcentrifuge tube containing 70% ethanol and stored at 4 °C for DNA extraction.

DNA extraction from blood and tick samples of bears
Blood and tick samples were collected to detect parasitic and tick-borne pathogens.Tick samples were dissected in sterilized 1 × PBS.For nymphs, the whole body was cut and manually homogenized, whereas each adult tick was cut into approximately two halves.Half of each adult tick sample was transferred to a clean microcentrifuge tube for DNA extraction.A total of 100 µL of sterile 1 × PBS was added to each tube and stored at 4 °C until DNA extraction.The other half was stored in 70% ethanol as a backup.DNA was extracted from blood and tick samples using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany), following the manufacturer's instructions.All DNA samples were eluted into a 100 µL final volume and stored at −20 °C until used for blood parasite and pathogen detection.

Sequencing and statistical analyses
PCR products were purified using a Plus DNA Clean/ Extraction Kit (GMbiolab Co., Ltd., Taichung, Taiwan) and sent for nucleotide sequencing (Genomics BioScience and Technology Co., Ltd., Taiwan).The sequence data were compared with known sequences deposited in the GenBank database using the National Center for Biotechnology Information (NCBI) nucleotide Basic Local Alignment Search Tool (BLAST).For genetic analysis, the validated sequences were aligned and analyzed using MegAlign (DNASTAR, Inc., Madison, WI, USA).All statistical analyses were performed using Graph-Pad Prism version 8.4.2.The numerical data of each blood parameter were summarized as medians, means, and standard deviations.Comparisons of blood parameters between each bear variable of the bears, such as sex, age, area, trap type, and pathogen occurrence, were performed using unpaired t-tests for parametric data and Mann-Whitney U tests for non-parametric data.Additionally, multiple comparisons of blood parameters among pathogen infection, co-infection, and noninfection statuses of the bears were performed using the Kruskal-Wallis test, and P < 0.05 was considered statistically significant.
The relationships between bear variables (sex, age, and area) and blood parasite and pathogen infection status; tick variables (sex, life stage, and area) and pathogen harboring status; and bear variables (sex, age, and occurrence of blood parasites and pathogens) and the presence of blood-parasite-and pathogen-carrying ticks were analyzed using the chi-square test.A two-tailed Fisher's exact test was used when the expected number of observations was less than five, and P < 0.05 was considered statistically significant.

Demography of captured Formosan black bears
In total, 21 Formosan black bears were successfully captured and released (14 in YNP and 7 in DSY) between November 2014 and July 2021.Among the bears captured, there were 13 adults (seven males and six females) and one male subadult in YNP and six adults (three males and three females) and one male subadult in DSY.Blood samples were successfully collected from all bears, and ectoparasites were collected from 13 bears (61.90%) (Additional file 1: Table S1).Additionally, five bears were captured by snares in 2014-2015, while the rest were captured by culvert traps in 2015-2021.

Hematological and plasma profiles of captured Formosan black bears
The hematological and plasma biochemical profiles of wild Formosan black bears are summarized in Tables 2  and 3. TPP, creatinine, Ca 2+ , Mg 2+ , and K + levels were higher in male bears than in females, and Fib value was higher in subadults than in adults.Additionally, ESR (1 h), ALP, triglycerides, and uric acid levels were higher in bears captured in YNP than in those captured in DSY, whereas the K + value was higher in bears captured in DSY.Moreover, WBC, segment, CK, and LDH values were higher in snare-captured bears than in culvert-captured bears, whereas PCV, thrombocytes, TPP, glucose and K + values were higher in culvert-captured bears.Regarding pathogen-infection-related differences, both ESR (30 min and 1 h) and globulin values were higher in infected bears than in non-infected bears, whereas albumin and A/G values were higher in non-infected bears.Furthermore, we compared blood parameters among bears infected with Babesia spp.(n = 2) and/or H. ursi (n = 7), co-infected with Babesia spp.and H. ursi (n = 6) and uninfected with the parasites (n = 6).Notably, there were significant differences in ESR (1 h; P = 0.0163), albumin (P = 0.0100), globulin (P = 0.0454), A/G (P = 0.0058), ALP (P = 0.0313), and Ca 2+ (P = 0.0121) values among groups (Tables 4, 5).Multiple comparisons showed that albumin and A/G values were higher in non-infected bears than in bears co-infected with Babesia spp.and H. ursi (P = 0.0077 and P = 0.0233, respectively).Additionally, Babesia-infected bears had significantly higher (P = 0.0453) globulin levels and lower (P = 0.0278) A/G value than uninfected bears.Moreover, ALP and Ca 2+ levels were higher in H. ursi-infected bears than in uninfected (P = 0.0327) and co-infected (P = 0.0314) bears (Table 5).

Blood parasite and pathogen detection in bears and ticks
Hepatozoon spp.gamonts were observed microscopically in thin blood smears (Fig. 3).Based on the morphology,    the beak-like protrusion at one end of the slightly curved gamont is one of the most characteristic morphological features of H. ursi.PCR showed that 13 (61.90%)bears harbored Hepatozoon DNA.Additionally, DNA fragments from 10 random Hepatozoon-positive samples showed 99.1-99.7%similarity with the H. ursi isolate (EU041718) listed in the GenBank database.Although no Babesia spp.were observed in thin blood smears, Babesia DNA fragments were detected in eight (38.10%)blood samples and showed 90.6-94.4% similarity with Babesia gibsoni (KF171473), Babesia microti (JX962779), Babesia odocoilei (KC460321), and Babesia spp.(KC465978).Additionally, H. ursi and Babesia spp.co-infection was observed in six (28.57%) bears (all captured in YNP).However, PCR showed that all blood samples tested negative for D. ursi, Bartonella spp., Rickettsia spp., and B. burgdorferi s.l.DNA.

H. ursi and Babesia spp. infection in Formosan black bears and their ticks
Notably, the prevalence rates of H. ursi and Babesia spp.infection in Formosan black bears were 61.90% (13/21) and 38.10% (8/21), respectively.Additionally, the prevalence of H. ursi infection in bears captured in YNP (92.85%) was significantly higher (P = 0.0001) than that in bears captured in DSY (0%); however, there was no significant difference in sex or age (Table 5).Moreover, there were no significant differences in the prevalence of Babesia spp.infection among bears of different sexes, ages, and areas (Table 7).
Furthermore, the prevalence rates of H. ursi and Babesia spp.infections in ticks collected from the bears were 65.41% (157/240) and 53.33% (128/240), respectively.Adult female ticks (73.84%) had a significantly higher (P = 0.0002) prevalence of H. ursi infection than adult male ticks (50.0%;Table 7).However, there were no significant differences in H. ursi infection rates among ticks from different locations or life stages.Additionally, ticks on bears captured in DSY (84.0%) had a significantly  7).However, there was no significant difference in Babesia infection rates among ticks of different sexes or life stages.

The relationships between Formosan black bears' status and their ticks carrying H. ursi and Babesia spp.
For each individual bear, the blood parasite infection status, numbers and species of ticks, and proportion of ticks harboring blood parasites are shown in Table 8.Ticks were collected from 13 of 21 (56.52%)bears.Notably, each bear was infested with up to four tick species per time (Table 6).H. flava was the predominant tick species (49.16%) infesting wild Formosan black bears in Taiwan, whereas the prevalence of the other tick species was below 20% (Table 4).Regardless of whether individual bears were infected with H. ursi and/or Babesia spp., the infesting ticks harbored blood parasites (Table 8).Ticks collected from Babesia-positive bears were more likely (P < 0.00001) to harbor Babesia spp.than those collected from Babesia-negative bears [65.30% (96/147) versus 34.40% (32/93)].However, there were no significant differences in H. ursi detection rates between ticks collected from H. ursi-positive and H. ursi-negative bears.

Discussion
To the best of our knowledge, this is the first study to establish a hematological and plasma biochemical database of free-ranging black bears in Taiwan and investigate ectoparasite infestation, blood parasites, and tick-borne pathogen.In this study, there were no significant differences in hematological values among the different sexes or ages.Notably, the median values of PCV, RBC, Hb, MCV, and other erythrocyte-related parameters in the present study and in wild American black bears were lower than those of captive Formosan black bears [13] and other Asian black bears [14], which may be due to a richer diet in captive environments [49,50].However, erythrocyte-related values in wild Formosan black bears were lower than those in wild American black bears [25,50].Overall, these differences in hematological values could be species-specific or caused by differences in food resources.
Additionally, plasma biochemical parameters were significantly affected by sex.For example, TPP, creatinine, Ca 2+ , Mg 2+ , and K + levels were significantly higher in male bears than in female bears; however, the reason for this remains unknown.TPP is an indicator of dietary protein levels and is correlated with the primary food sources of bears [51].Additionally, the lower levels of Ca 2+ in female bears than in male bears may be attributed to long-term pregnancy and lactation [50,52].In the present study, pathogen-infected bears had higher levels of inflammatory indicators, including ESR (30 min and 1 h) and globulin than uninfected bears.Moreover, there were significant differences in ESR (1 h), albumin, globulin, A/G, ALP, and Ca 2+ levels between uninfected and pathogen-infected bears.Furthermore, Wright-Giemsa staining indicated the presence of Hepatozoon spp.gamonts in the blood of bears with high ESR and globulin values.Notably, changes in albumin, globulin, and ALP levels have also observed in other Babesiainfected hosts, such as camels (Camelus dromedarius) [53], dogs (Canis familiaris) [54], and cattle [55] as well as Hepatozoon-infected dogs [56][57][58].Collectively, these hematological and plasma data may serve as a reference for future investigation on host and tick-borne pathogen relationships in wild bears.
Importantly, the differences in capture procedures also seemed to affect the blood profile of the bears.Consistent with previous findings in wild European brown bears and American black bears [28,59], WBC, segments, CK, and LDH values were elevated in snare-captured bears, which may be attributed to potential stress and muscle injuries.However, diagnosing muscle injuries in bears requires physical examinations and imaging, which can be challenging in the field.Information based on satellite tracking and blood testing results seemingly can only  Therefore, we suggest that, when circumstances warrant, traps should be considered as a priority.Otherwise, snare traps should only be applied in remote areas with poor accessibility or emergency conditions, and when traps can be monitored in real time or checked daily.Currently, 5 species of soft ticks and 44 species of hard ticks have been recorded in Taiwan [60][61][62].Among the eight species of hard ticks found in wild Formosan black bears in this study, only A. javanense has not been previously recorded in Taiwan.H. flava appears to be the predominant tick infesting wild Formosan black bears, and has also been found in several mammals in Taiwan, including wild boars (Sus scrofa), deer (Rusa unicolor), and dogs [63].Additionally, H. flava, A. testudinarium, H. longicornis, and D. taiwanensis have been recorded in wild Japanese black bears (U. t. japanicus) [17,23].A. javanense ticks are commonly found in reptiles and mammals, especially in pangolins [64].Phylogenetic analysis and data of tick species in other animals in the same area would be helpful in investigating the distribution and sources of A. javanense in the country.
To the best of our knowledge, this is the first study to report H. ursi and Babesia spp. in wild Formosan black bears and their ticks.Notably, H. ursi infection had a lower prevalence in Formosan black bears (61.90%) than in Japanese black bears (76.3% and 100%) [17,43], Indian sloth bears (Melursus ursinus; 70%) [18], and Turkish brown bears (Ursus arctos; 100%) [65].Additionally, H. ursi detected in this study had a 99.1-99.7%nucleotide similarity with isolates from Japanese black bears (EU041718).Currently, H. ursi is the only Hepatozoon spp.detected in the family Ursidae [66].Although wild Asiatic black bears have high H. ursi prevalence, there are no current reports on Hepatozoon spp. in other bear species such as American black bears, European brown bears, and polar bears [42,67,68], which may be because the suspected vector, H. flava, is mainly distributed in Asian countries [17,[69][70][71][72].
Hepatozoon spp.can be transmitted to hosts via ingestion of sporulated oocysts in ticks or other arthropod vectors [73][74][75].In addition to the ingestion of vectors, predators, such as wild canids that hunt grey squirrels harboring the cystozoite stage of Hepatozoon spp., have also been suggested as a possible transmission route [76][77][78].Hepatozoon infection in wild carnivores, such as the red fox (Vulpes vulpes) [79], European wild cat (Felis silvestris silvestris) [80], and pine martens (Martes martes) [81], usually cause little harm to the host.However, Hepatozoon-infected wildlife, which play an important role in the circulation of this pathogen, may represent a complementary natural reservoir for domestic animals [82].For example, immature and mature meronts of Hepatozoon were histopathologically identified in the lungs of Hepatozooninfected Japanese black bears [66].Additionally, the lungs of these bears may harbor the schizogonic developmental stages of H. ursi [17].
Infections with Babesia spp.have been previously reported in Japanese and American black bears [43,[83][84][85].In the present study, Babesia spp.infecting Formosan black bears were closely related to other Babesia species, such as B. gibsoni from dogs, B. microti from foxes (Vulpes spp.), and B. odocoilei from elks (Cervus elaphus canadensis).Similarly, Babesia DNA from American black bears was closely related to sequences from raccoons or domestic dogs [83].Collectively, these results indicate that black bears can be infected by several Babesia spp.Furthermore, the unidentified Babesia spp.found in this study showed a 90.6-95.5% sequence similarity with known Babesia spp., highlighting the importance of conducting additional amplification and sequencing of the full-length 18S rRNA gene or other specific genes of Babesia to achieve a more detailed genetic characterization.Babesia spp.are commonly transmitted to hosts by competent feeding ticks, followed by transfusion of infected blood products and vertical transmission [86,87].Babesia infections are typically asymptomatic in wild animals [88,89].For example, the clinical impact of babesiosis in bears is limited, and only a single episode of anemia has been reported in a Babesia-infected Japanese brown bear (U. t. japonicus) heavily infested with ticks [90].However, the role of black bears as potential wildlife reservoirs of Babesia and other vector-borne pathogens warrants further investigation.
Although sex, age, or origin did not significantly affect the prevalence of blood parasites related to sex, age, or location, the prevalence of H. ursi infection was significantly higher in bears captured in YNP (13/14; 92.85%) than in those captured in DSY (0/7; 0%).H. ursi seemed to dynamically circulate among ticks, bears, and the environment in the YNP, but not in the DSY.Among the tick species that harbored Hepatozoon spp., H. flava is likely the most epidemiologically relevant.In Japan, mature Hepatozoon oocysts have been found in H. flava and H. japonica collected from dead Japanese black bears [17].Overall, these findings indicate that H. flava plays an important role in Hepatozoon transmission to wild black bears.However, the vector competence of H. flava for Hepatozoon transmission requires further study.In the present study, female ticks had a higher prevalence of H. ursi infection than male ticks.Unlike male ticks, female ticks have a larger physiological demand for blood because they require it for engorgement prior to egg laying.This engorgement process provides them with additional opportunities to acquire pathogens from the host.
In Taiwan, Dermacentor taiwanensis and H. flava show a high prevalence of Babesia infections.Dermacentor taiwanensis infests several wild animals, including boar (Sus scrofa taivanus), bamboo partridge (Bambusicola thoracica), murine rodents (Bandicota, Rattus, Mus), tree squirrel (Callosciurus), hares (Lepus), and mustelid and viverrid carnivores (Mustela, Melogale, Paguma) [91].In the present study, BLAST analysis revealed that Babesia DNA samples from ticks matched several species of Babesia.Although the species of Babesia was not identified due to the wide host range of ticks, we showed that ticks that infest Formosan black bears may be infected by several species of Babesia.Additionally, ticks on bears captured in DSY were more likely to harbor Babesia spp.than those captured in YNP.To further clarify whether the location affects the prevalence of Babesia infection in vectors, Babesia should be detected in ticks from the same environment.
Notably, regardless of whether the individual bears were infected with H. ursi and/or Babesia spp., collected ticks harbored blood parasites.Additionally, ticks collected from Babesia-positive bears were more likely to harbor Babesia spp.than ticks collected from Babesianegative individuals.Importantly, the collected ticks harbored blood parasites even in bears that were not infected for several reasons, such as meal contamination (current or previous blood meals) and environmental contamination (on tick or host surfaces) [92].Bears and ticks inhabit environments with a high prevalence of blood parasites, allowing ticks to carry these blood parasites [92].Considering that previous research on Babesia spp.has rarely mentioned the relationship between host infection status and infesting vectors, this finding provides information on the association between Babesia spp.-infected ticks and Formosan black bears.Although only H. ursi and Babesia spp.were detected in wild Formosan black bears and in infesting ticks in this study, the other investigated blood parasites and pathogens such as D. ursi, Bartonella spp., Rickettsia spp., B. burgdorferi s.l., Ehrlichia chaffeensis, Anaplasma phagocytophilum, and Toxoplasma gondii have been reported in Japanese and American black bears [16,20,26,93,94].Overall, the different results can be attributed to various factors, including geographical location, host specificity, tick species diversity, or sampling methodology.Comprehensive epidemiological surveys of parasites and other pathogens, particularly of endangered species, are recommended.
Amblyomma testudinarium, I. acutitarsus, and Rhipicephalus sanguineus s.l.ticks are frequently found in humans in Taiwan, followed by H. hystricis and Rhipicephalus haemaphysaloides [95][96][97].These ticks harbor and act as potential vectors for zoonotic and human pathogens.In November 2019, a single human case of "severe fever with thrombocytopenia syndrome" (SFTS) was reported in Taiwan, resulting in death 40 days after the onset of clinical signs [98].The tick-borne SFTS virus can be transmitted by H. longicornis [99], and has also been detected in A. testudinarium in Korea [100].In Taiwan, the SFTS virus has been identified in serum samples collected from sheep, cattle, and dogs, and in R. microplus ticks collected from cattle [101].Additionally, the Oz virus, a novel Thogotovirus that causes febrile illness and death in humans, was first isolated from A. testudinarium in Japan [102].Overall, these findings emphasize the potential risk of zoonotic pathogen transmission.In the present study, both H. longicornis and A. testudinarium infested Formosan black bears.Additionally, both species may potentially infest humans and increase the risk of zoonotic diseases, such as SFTS and febrile illness caused by the Oz virus in Taiwan.

Conclusions
In this study, we established a database of the hematological and plasma profiles of free-ranging Formosan black bears in Taiwan and investigated the occurrence of ectoparasites, blood parasites, and vector-borne pathogens to elucidate the health status and pathogen dynamics within the bear population in Taiwan.Notably, a regional hematological and plasma biochemical database across various subspecies of Asiatic black bears in 18 countries [103] may serve as an essential resource for wildlife veterinarians and biologists in disease diagnosis and monitoring.
1031700938 and 1041701149) and the National Pingtung University of Science and Technology Laboratory Animal Care and Use Committee (IACUC No. NPUST-102-051 and NPUST-104-047).

Fig. 1
Fig. 1 Two study sites of Formosan black bears (Ursus thibetanus formosanus) captured and released during 2014-2021-Yushan National Park (yellow line) and Daxueshan National Forest Recreation Area (blue line).The sites are distributed in the Central Mountain Range and Xueshan Mountain Range, respectively, of Taiwan.Red grids (1 × 1 km 2 ) represent the predicted distribution of Formosan black bears[29]

Fig. 2
Fig. 2 The setting of (a) snare and (b) culvert traps and sample collection for (c) subadult and (d) adult Formosan black bears

Fig. 3
Fig.3The beak-like protrusion at one end of the slightly curved gamont of H. ursi (arrowed) found in thin blood smears of Formosan black bears

Table 1
Primers for detecting blood parasites and pathogens of wild Formosan black bears used in this study

Table 2
Hematological profiles and the variation by bear sex, age, captured area, trap type, and occurrence of pathogens in wild Formosan black bears

Table 3
Plasma biochemical profiles and the variation by bear sex, age, captured area, trap type, and occurrence of pathogens in wild Formosan black bears N/A, Not applicable; SD, standard deviation * Significant values (p < 0.05) are marked with bold font

Table 4
Comparison of hematological profiles and pathogen occurrence variation in wild Formosan black bears

Table 5
Comparison of plasma biochemical profiles and pathogen occurrence variation in wild Formosan black bears Among rows: the values with different superscript letters in a row are significantly different (P < 0.05)

Table 6
Numbers of ticks in different species collected from Formosan black bears and the proportion of ticks that tested PCRpositive for blood parasites

Table 7
Numbers of H. ursi-and Babesia spp.-PCR-positive samples in different variables of wild Formosan black bears and ticks

Table 8
Numbers of ticks infesting bears, diversity of ticks, and H. ursi and Babesia spp.detected in wild Formosan black bears and ticks PCR-positive; −, PCR-negative * +,